We have begun a project to establish an in vitro transcription system that recapitulates the activation of PvpsL, the promoter for the V. cholera vpsL gene needed for biofilm formation, in the presence of RNA polymerase, VpsR, and c-di-GMP. Using this system, we aim to determine the step(s) in transcription initiation that requires VpsR/c-di-GMP for activation, to determine the protein-protein and protein-DNA interactions needed for the activated complex, to construct a molecular map of VpsR/c-di-GMP/RNAP/DNA in the active transcription complex, and to investigate c-di-GMPs role in VpsR structure and function.